For many years our research program has been focused on the mechanisms of gene regulation in early sea urchin embryos, and this will continue to be its major thrust. We have developed and successfully exploited an excellent suite of technologies for complete structure/function analysis of embryonic cis-regulatory systems. Among these technologies are: a gene transfer procedure that permits expression on many groups of hundreds of embryos at a time, so that the spatial and temporal behavior of expression vectors bearing mutations or consisting in part of synthetic DNAs can be studied in great variety; a novel methodology for knocking our transcription factor activity under experimental genetic control; a methodology for cloning transcription factors after affinity purification; use of large-scale high density arrayed clone libraries; advanced imaging technology for digital recording of spatial expression of fluorescent receptors; and a complete armamentarium of lineage tracing, cell biological, microdissection and in situ procedures for assay of spatial gene expression. In the next period we intend to extend and deepen our mechanistic understanding of gene regulation in early development, by exploiting these methods to characterize functionally some additional cis-regulatory elements that lie in key positions in the genetic control network of the early embryo. We will complete our analysis of the complex Endo16 cis-regulatory system, identifying specific repressors and activators that are not yet characterized. Some additional differentially expressed, downstream genes isolated by differential screening of high density arrayed libraries will be subjected to similar analysis, to determined their cis-regulatory organization; and to identify positive and negative spatial regulatory interactions & elements that lie at the termini of signal transduction systems. A major effort will be directed to cis-regulatory analysis of the systems controlling early zygotic expression of regulatory genes encoding transcription factors. Among these will be factors already identified as controllers of the terminal genes we have already studied. Our overall objective is to understand the gene regulatory network that is set into operation as control is shifted from the maternal to the zygotic transcription regulatory apparatus in cleavage and blastula stages. We will utilize several new approaches to isolate gene batteries, i.e., sets of downstream genes that are controlled by the same set of transcription factors. Major efforts will be devoted to trying to understand the disposition and complexity of maternal transcription factors, the functions of which are known from cis-regulatory analysis of their target genes. Here we have three specific objectives; first, localization in the early embryo by conventional confocal immunocytology, and assessment of regional activity in respect to DNA target site binding by a microcapillary-based shift procedure that we developed; second, analysis of transcription factor variants by mass spectrometry and other methods; and third, localization of variants in mass-isolated cell fractions representing different territories of the early embryos. We are also planning to apply our gene regulatory approaches to the significance of cell contacts in the blastula wall. The synthesis and turnover rates of beta-catenin will be measured to determine whether control of its availability is maternal or zygotic in the cleavage-blastula-stage embryo, and if zygotic we will investigate its cis-regulatory system. We will also study the mechanistic basis on CyIIIa cytoskeletal actin gene down-regulation in disaggregated ectoderm cells, exploiting our complete understanding of the CyIIIa cis- regulatory system. An additional approach to functional significance in cis- regulatory systems is to use interphyletic gene transfer. Thus we will determine the locus of expression in sea urchin embryos of ascidian cis- regulatory systems that control early expression of key transcription factors in endoderm (GATA factors, some homeodomain factors, and forkhead factors); and in mesoderm (e.g., snail and not). Similarly we will provide the corresponding sea urchin cis-regulatory reporter systems for tests in ascidian embryos. Since transcription factors can be directly isolated from sea urchin extracts, this approach may provide molecular evidence of the upstream (often maternal) regulators that generate early zygotic regulatory gene expression in chordate embryos as well.